GDF-5 is a member of the Bone Morphogenetic Proteins (BMP), which is a subclass of the TGF-β superfamily of proteins. GDF-5 includes several variants and mutants, including mGDF-5 first isolated from the mouse by Lee (U.S. Pat. No. 5,801,014). Other variants include MP52, which is the patented name (WO 95/04819) for the human form of GDF-5, which is also known as hGDF-5 and also as LAP-4 (Triantfilou, et al. Nature Immunology 2, 338-345 (2001)); also CDMP-1, an allelic protein variant of hGDF-5 (WO 96/14335); also rhGDF-5, the recombinant human form manufactured in bacteria (EP 0955313); also rhGDF-5-Ala83, a monomeric variant of rhGDF-5; also BMP-14, a collective term for hGDF-5/CDMP-1 like proteins; also Radotermin, the international non-proprietary name designated by the World Health Organization; also HMW MP52's, high molecular weight protein variants of MP52; also C465A, a monomeric version wherein the cysteine residue responsible for the intermolecular cross-link is substituted with alanine; also other active monomers and single amino acid substitution mutants including N445T, L441P, R438L, and R438K. For the purposes of this application the term “GDF-5” is meant to include all variants and mutants of the GDF-5 protein, wherein rhGDF-5 is the exemplary member having 119 amino acids.
All members of the BMP family share common structural features including a carboxy terminal active domain and share a highly conserved pattern of cysteine residues that create three intramolecular disulfide bonds and one intermolecular disulfide bond. The active form can be either a disulfide-bonded homodimer of a single family member or a heterodimer of two different members (see Massague, et al. Annual Review of Cell Biology 6:957 (1990); Sampath, et al. Journal of Biological Chemistry 265:13198 (1990); Celeste et al. PNAS 87:9843-47 (1990); U.S. Pat. No. 5,011,691, and U.S. Pat. No. 5,266,683). The proper folding of the GDF-5 protein and formation of these disulfide bonds are essential to biological functioning, and misfolding leads to inactive aggregates and cleaved fragments.
The production of BMP's from genetically modified bacteria, and of GDF-5 in particular, utilizes plasmid vectors to transform E. coli to produce monomer GDF-5 protein in high yield (see for example Hotten U.S. Pat. No. 6,764,994 and Makishima U.S. Pat. No. 7,235,527). The monomer is obtained from inclusion bodies, purified, and refolded into homodimers of GDF-5 protein to produce the biologically active dimer of the GDF-5 protein. The steps leading to the dimer utilize various pharmaceutically unacceptable materials to modify the solubility in order to enable the separation and purification of the GDF-5 protein.
The degradation of proteins in general has been well described in the literature, but the storage and solubility of bone morphogenetic proteins, particularly GDF-5 has not been well described. BMP-2 is readily soluble at concentrations greater than 1 mg/ml when the pH is below 6, and above pH 6 the solubility can be increased by the addition of 1 M NaCl, 30% isopropanol, or 0.1 mM heparin (Ruppert, et al. Eur J Biochem 237, 295-302 (1996). GDF-5 is nearly insoluble in physiological pH ranges and buffers and is only soluble in water at extreme pH (Honda, et al. Journal of Bioscience and Bioengineering 89(6), 582-589 (2000)). GDF-5 is soluble at an alkaline pH of about 9.5 to 12.0, however proteins degrade quickly under these conditions and thus acidic conditions are used for the preparation of GDF-5 protein.
Biocompatible compositions of the GDF-5 protein present great challenges to obtain reasonable solubility and concurrent stability of the protein. The current method of storage for GDF-5 protein utilizes 10 mM HCl at pH 2 and −80° C. for long-term storage, but even these conditions provide for some degradation of the protein, particularly with repeated freeze-thaw cycles. We performed a trypsin digestion of late eluting species of the GDF-5 protein and found non-tryptic peptide fragments using MALDI-TOF (matrix assisted laser desorption ionization-time of flight mass spectrometry) analysis, indicating acid-catalyzed cleavage of the protein during storage and subsequent aggregation of the fragments. We also separately performed sequential freeze-thaw cycles and prolonged exposure to elevated temperatures of GDF-5 protein solutions. Both of these tests showed degradation of the protein in the current 10 mM HCl storage solvent. Thus there is a need for improved compositions for the handling and storage of GDF-5 protein solutions.